Major Histocompatibility Complex (MHC) class I molecules bind peptides (epitopes) within the cell and transport them to the plasma membrane for inspection by CD8+ T lymphocytes (TCD8+). Upon recognition of the peptide, T CD8+ respond by killing the peptide-presenting cell and/or releasing an array of soluble mediators that drive protective responses. Such responses are the basis for viral clearance, many autoimmune responses, and successful cancer immunotherapy. For many years we have been interested in the cellular processes that generate class I-binding peptides. Despite the existence of an outline of antigen processing that has gained general acceptance, several fundamental areas remain uncertain. These are: 1) The forces that drive antigen into the processing pathway. Our established results are consistent with a model, distinct from the generally accepted "DRiP" (defective ribosomal product) model, in which antigen is mainly targeted for degradation prior to folding and quality control decisions. The degree to which proteins that have interacted with the quality control machinery supplement this primary epitope supply will depend upon inherent stability. Using a variety of approaches we will test several key aspects of this model. 2) That role of ubiquitinylation (Ub'n) in targeting antigen for processing. Until recently, it was assumed that Ub'n is essential for the turnover of most cytosolic proteins and the presentation of most antigens. Now, many alternative modes of degradation are known and the participation of Ub in protein breakdown and antigen presentation is far from predictable. Through the use of a superexpressed "dominant negative" Ub molecule in combination with sophisticated techniques for peptide analysis we will examine the global impact of Ub'n on epitope supply in an effort to develop general rules. 3) Generation of the C-terminus of the epitope. This is presumed to be the exclusive role of the proteasome but our preliminary data strongly suggest that in some cases, the C-terminus can be generated by a non- proteasomal cytosolic protease. Our aims are to better understand the specificity of this activity, to identify its source, and determine the scope of its influence upon antigen processing. Clarifying these key aspects of MHC class I antigen processing will provide more informed approaches to vaccine design, treatments of autoimmune diseases, and cancer immunotherapy. The overall goal of this work is to understand how antigens are targeted for MHC class I-restricted antigen processing. The cells of the immune system that are triggered by MHC class I-restricted antigen presentation, CD8+ T cells, plays a key role in limiting the spread of acute viral infections and in keeping chronic/latent viral infections in check and can be very effective against cancers . We anticipate that information gained from the proposed research plan will point the way to new strategies in vaccine design for maximizing the activities of CD8+ T cells.